The present invention relates to a method and apparatus which facilitates the incubation of samples at several different temperatures in a cyclical program. More particularly, the invention is directed to a process and equipment for carrying out a DNA amplification through replication of a stretch of a particular sequence of DNA or RNA, employing polymerases.
The present invention finds utility in the "polymerase chain reaction" (PCR) described, for example, in U.S. Pat. No. 4,683,202, and in which a stretch of DNA is copied using a polymerase. The general procedure there disclosed is to anneal a piece of primer DNA (oligonucleotide) at a temperature T1, to any stretch of single-stranded DNA (template) with a complimentary sequence. The DNA polymerase copies the primed piece of DNA at a temperature T2. At a temperature T3, the newly copied DNA and the primer dissociate from the template DNA, regenerating single-stranded DNA. As the cycle is caused to repeat itself, the temperature returns to T1 and the primer attaches itself to any strand of single-stranded DNA with complimentary sequence, including the ones just recently synthesized.
The procedure described produces any particular nucleic acid sequence from a given sequence of DNA in amounts which are substantially increased with reference to the amount initially present, thus facilitating detection of the nucleic acid sequences involved. Thus, the method described obviates the difficulties of detecting the presence of the DNA sequence using labeled oligonucleotide probes. The method employed in the practice of the invention effects a synthesis of the nucleic acids from an existing sequence, thus producing significantly increased amounts of a given nucleic acid of a completely specified sequence.
As described above, the replication and amplification of a particular sequence of DNA by means of the PCR technique requires utilization of a temperature profile. For efficient functioning of the PCR process, precise control of temperature at each stage of the cycle is essential. Moreover, it is important that each temperature (that is, T1 through TN) is reached as quickly as possible without exceeding the set point (that is, without "overshooting"). These goals have not been achieved or optimized in prior art techniques. One such technique is to utilize three water baths at temperatures T1, T2 and T3, between which the sample-containing tubes are cycled, either manually or automatically. In another method, electrically heated metal blocks are used. The water bath procedure has proved to be cumbersome and difficult to automate. Moreover, the heat transfer to the sample tubes has not proved to be particularly efficient. In addition, the temperature existing inside the tubes is not easily measured on a continuous basis.
The electrically heated metal block, while less cumbersome mechanically, also has problems, particularly such as related to the heat capacity of the metal. In this system, it is difficult to effect rapid changes in the temperature of the metal block, and thus the time for each cycle is unduly lengthened. In addition, the heat transfer to the liquid inside the sample tubes varies with the metal/tube surface area. Any reduction in contact area (poor fit, dust, or foreign matter between the tube and the block) causes the temperature in the tube to mismatch with that of the metal block within the treating period.
It is, therefore, a principal aim of the present invention to obviate shortcomings of prior art methods and techniques and to provide a system in which cyclical changes in temperatures are more effectively controlled, transition from one temperature to another is achieved more rapidly and more expeditiously, and in which there is a more precise correlation between the temperature of the treating environment and the temperature of the solutions in the sample-containing tubes or vials. The present invention ensures efficient heat transfer, good temperature control, and provides practical techniques for continuous monitoring of the temperature in the samples being treated.